SUMMARY Ribosome rescue pathways are conserved throughout bacteria, but the reason these pathways are important for physiology is not understood. The long-term goal of this project is to understand the function of ribosome rescue pathways and to target these pathways for new antibiotics. The overall objective of the proposed project is to use ribosome rescue inhibitors as probes to understand ribosome rescue at the atomic, molecular, and cellular levels. The central hypothesis of this work is that ribosome rescue is universally required for maintaining protein synthesis capacity in bacteria and that inhibitors of rescue target conserved components of the translation machinery. The rationale for pursuing the proposed research is that it will determine why ribosome rescue is conserved in bacteria and will enable development of new antibiotics. The central hypothesis will be tested by pursuing the following specific aims: 1) identify the molecular interactions required for ribosome rescue, 2) determine why ribosome rescue is important for bacterial physiology and 3) discover new mechanisms for ribosome rescue. Published work and preliminary data have identified four chemically related families of small molecules that inhibit ribosome rescue. These compounds will be used in Aim 1 to a) identify the molecular targets and binding sites responsible for inhibiting ribosome rescue and b) determine how interactions with target molecules block ribosome rescue but not translation. We will use the small molecule inhibitors as chemical biology tools to examine three outcomes from inhibition of ribosome rescue: the status of ribosomes, which are the direct target of ribosome rescue; and the proteomic and transcriptomic responses when ribosome rescue becomes limiting for growth. The working hypothesis for Aim 3 is that at least one ribosome rescue system is required in all bacteria. This hypothesis will be tested by using Tn-seq to identify alternative ribosome rescue factors in Bacillus subtilis and Francisella tularensis, the only species shown to survive without tmRNA that do not contain one of the known backup systems. The use of small molecule inhibitors for chemical biology experiments to probe ribosome rescue is highly innovative, and the work proposed here is significant because it will delineate the physiological requirement for ribosome rescue pathways in bacteria and identify how these pathways can be inhibited.